Glucokinase is a key regulator of glucose homeostasis in the human body. Dysfunction in the regulation and/or activity of glucokinase causes a diverse range of diseases including maturity onset diabetes of the young (MODY) and hyperinsulinemia of infancy (HI). Human glucokinase is a monomeric enzyme that is allosterically regulated by its sugar substrate, D- glucose. This kinetic cooperativity is critical to maintaining proper glucose serum levels in vivo. The goal of this study is to elucidate the mechanistic basis for cooperativity in human glucokinase. The experiments described herein combine a powerful genetic selection system for human glucokinase activity with mechanistic enzymology to provide a kinetic and molecular description of the conformational transitions that give rise to cooperativity. The specific aims are: (1) To determine the mechanism of activation of hyperinsulinemia-linked glk mutations; (2) To investigate conformational heterogeneity in unliganded glucokinase via chemical quench-flow techniques; (3) To investigate the role of a secondary structural allosteric switch in glucokinase cooperativity; (4) To determine whether prolyl cis/trans isomerization is responsible for the slow interconversion of enzyme conformations. Achieving the objectives outlined in this application promises to impact the development of future glucokinase-targeted therapeutics for diabetes and hyperinsulinemia. PUBLIC HEALTH RELEVANCE: In the past five years, human glucokinase has emerged as an attractive therapeutic target for diabetes. Numerous small-molecule activators of glucokinase have been identified and several have been shown to be effective at modulating blood glucose levels in animal models. The successful completion of the aims outlined in this application will provide critical new information about the mechanisms of glucokinase regulation in vivo. This information has the potential to significantly impact future efforts to design glucokinase-targeted therapeutics for diabetes and hyperinsulinemia.